作者希望感谢 p. Karagiannis 博士阅读论文和好友教授的技术支持。

这里描述的所有方法都得到了当地道德委员会 (同志社大学伦理委员会: 15033) 的批准, 严格按照《赫尔辛基宣言》规定的标准。所有与会者都获得了知情同意。参加者为男性休闲运动员 (年龄, 20.1 岁, 1.2 岁; 体重, 69.7 英寸6.2 公斤; 身高, 176.1 厘米5.8 公分; 身体脂肪 13.6 @ 1.5% 和 VO2最大, 52.6 @ 6.6 毫升/分钟1/千克1)。
1. 制定《行动议定书》
注: 本研究包括 HIIE 协议中 antecubital 静脉采血对血液激素和代谢物动态的评价。
<ol><li>要求该主题进行热身与5分钟的自行车在测力计低强度。热身后, 让主题休息5分钟。</li>
	<li>要求主题执行以下 HIIE 在腿周期测力计: 三回合三十年代高强度锻炼, 分离由4分钟休息 (图 1)。 注: 三十年代回合应设置在强度等于先前执行的盖特试验的功率平均值的90%。有关盖特测试的完整说明, 请参阅其他8 。</li>
	<li>要计算三十年代回合的工作量, 请使用以下公式: 工作 = 力量 x 距离 (等式 1) 力 = 质量 x 加速度 (等式 2) 工作 = (质量 x 加速度) x 距离 (等式 3) 注意: 在这个模型中, 质量是工作负载 (千克), 加速度为9.81 米/秒2。距离是轮子直径 (1.622 m)。由于车轮旋转3.7 定时/旋转, 每踏板旋转的距离是 3.7 x 1.662。此外, 因为踏板频率是90转速/分钟1, 我们必须乘以一个踏板距离90。 工作负载 x 9.81 x 3.7 x 1.622 x 90 (等式 4) 工作负荷 = 工作/(9.81 x 3.7 x 1.622 x 90) (等式 5)</li>
</ol>2. 导尿管
注意: 应预先标记管子和 vacutainers, 以避免丢失时间。
<ol><li>将止血带放在选择插管的手臂上。要做到这一点, 在选择的手臂后面放置一个弹性带。伸展乐队, 做一个环与乐队周围的手臂和领带的弹性。</li>
	<li>选择可见的 antecubital 静脉, 用乙醇或酒精消毒皮肤。</li>
	<li>将消毒套管的尖端插入静脉, 并将其与皮肤的一个近似角度。 注: 套管应插入从近端到远端方向。如果插管成功, 应该观察反冲的血液。</li>
	<li>用手固定套管, 轻轻取出针。 注意: 为了保持针在运动过程中的角度, 棉组织可以在皮肤和针头之间使用。用磁带修复此结构。</li>
	<li>连接一个 vacutainer 与肝素和吸入的第一个血液样本 (约2.5 毫升)。紧接着, 把血液样本放在一个冰盒里, 不超过40分钟。如果要分析血液中的气体, 请向助手求助。</li>
	<li>将管连接至导管, 并将肝素注射至管腔全长, 以避免凝血。把蝴蝶关上</li>
	<li>使用胶带, 稳定导管。确保在这一点上, 主体的手臂移动不会干扰血液的收集。要确认此情况, 请确保针位置不受任何运动的影响。</li>
</ol>3. 血液抽样
<ol><li>使用 vacutainer 除去肝素, 直到导管内只有血液。当观察到血液闪回时, 收集样本如步骤2.5 所述。</li>
	<li>标本采集后, 再将肝素注入导管内, 以避免凝血。在此操作之后, 可以重复执行多个示例集合中的步骤2.5。</li>
	<li>最后采集样品后, 小心取出插管管, 用酒精清洗皮肤。为避免可能出血, 请要求该主体在插管的地方保持消毒棉。</li>
	<li>在 vacutainer 管中采集血液后, 小心地将大约1.5 毫升的血液传递给1.5 毫升管, 并将管子放在冰上。</li>
	<li>用乳酸检测仪测量剩余血液中的乳酸。在同一天, 获得血浆的血液样本 (约1.5 毫升的血液)。</li>
	<li>为血浆分离, 离心机血液样品为5分钟在 5000 x g 在 4 oC, 并且分离上清在一个新的1.5 毫升管子。</li>
	<li>重复离心一次, 净化等离子。</li>
	<li>血浆分离后, 将样品保存在-80 oC, 直到荷尔蒙分析。</li>
	<li>根据制造商的协议, 使用 ELISA 试剂盒测量血浆胰岛素 (请参阅材料表)。</li>
</ol>

<ol>
	<li>Wahl, P., Mathes, S., K&#246;hler, K., Achtzehn, S., Bloch, W., Mester, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+active+vs.+passive+recovery+during+Wingate-based+training+on+the+acute+hormonal,+metabolic+and+psychological+response.">Effects of active vs. passive recovery during Wingate-based training on the acute hormonal, metabolic and psychological response.</a> Growth Horm IGF Res. 23 (6), 201-208 (2013).</li><li>Wahl, P., Mathes, S., Achtzehn, S., Bloch, W., Mester, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+vs.+passive+recovery+during+high-intensity+training+influences+hormonal+response.">Active vs. passive recovery during high-intensity training influences hormonal response.</a> Int J Sports Med. 35 (7), 583-589 (2014).</li><li>Nalbandian, H. M., Radak, Z., Takeda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+active+recovery+during+interval+training+on+plasma+catecholamines+and+insulin.">Effects of active recovery during interval training on plasma catecholamines and insulin.</a> J Sports Med Phys Fitness. , (2017).</li><li>Stolen, T., Chamari, K., Castagna, C., Wislff, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+Soccer.">Physiology of Soccer.</a> Sport Med. 35 (6), 501-536 (2005).</li><li>Bangsbo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology+of+soccer--with+special+reference+to+intense+intermittent+exercise.">The physiology of soccer--with special reference to intense intermittent exercise.</a> Acta Physiol Scand Suppl. 619, 1-155 (1994).</li><li>MacInnis, M. J., Gibala, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+adaptations+to+interval+training+and+the+role+of+exercise+intensity.">Physiological adaptations to interval training and the role of exercise intensity.</a> J Physiol. 595 (9), 2915-2930 (2017).</li><li>Emberts, T., Porcari, J., Dobers-Tein, S., Steffen, J., Foster, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+intensity+and+energy+expenditure+of+a+tabata+workout.">Exercise intensity and energy expenditure of a tabata workout.</a> J Sports Sci Med. 12 (3), 612-613 (2013).</li><li>Smith, J. C., Hill, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+energy+systems+during+a+Wingate+power+test.">Contribution of energy systems during a Wingate power test.</a> Br J Sports Med. 25 (4), 196-199 (1991).</li><li>Dotan, R., Bar-Or, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Load+optimization+for+the+Wingate+Anaerobic+Test.">Load optimization for the Wingate Anaerobic Test.</a> Eur J Appl Physiol Occup Physiol. 51 (3), 409-417 (1983).</li><li>Carrasco-Benso, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adipose+tissue+expresses+intrinsic+circadian+rhythm+in+insulin+sensitivity.">Human adipose tissue expresses intrinsic circadian rhythm in insulin sensitivity.</a> FASEB J. 30 (9), 3117-3123 (2016).</li><li>Hatfield, D. L., Nicoll, J. X., Kraemer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Circadian+Rhythm+on+Power,+Force,+and+Hormonal+Response+in+Young+Men.">Effects of Circadian Rhythm on Power, Force, and Hormonal Response in Young Men.</a> J Strength Cond Res. 30 (3), 725-732 (2016).</li></ol>

作者没有什么可透露的。

在本研究中, antecubital 静脉插管, 在高间隔运动中成功采集血液标本。以此方法为例, 对血浆胰岛素的血乳酸和血浆分离后的血液标本进行了分析。这种方法的主要优点是它允许在短时间内采集血样。同时, 它也有一些局限性。例如, 允许使用的有限的锻炼方式;需要的助理人数, 以及所需的专门培训人员;和低样本量的风险, 由于在前臂的低血压。
对于一个成功的血样采集, 该主体的手臂应处于静态位置。由于前臂的移动性降低, 其目前的形式可以仅适用于少数练习模式。在这里, 我们描述了如何应用该方法时, 演习是在一个周期测力计, 但随着修改, 它可以在跑步机上使用。在这两种情况下, 都需要有血液收集经验的人员。
关于《行动议定书》, 有一些考虑应予解决。如果要对几个试验进行评估 (例如, 在两次回合之间进行和不进行主动恢复), 则测试的决定时间至关重要。为了避免日荷尔蒙变化10,11, 建议在不同的时间在同一时间执行所有试验。此外, 新陈代谢的影响, 每天的时间和膳食消耗。因此, 在试验前建议禁食。此外, 为了提高重现性, 建议在测试之前, 由受试者使用练习协议的熟悉期。在这一时期, 实验对象应进行 (逐步) 类似的运动的主要测试。
最后, 我们描述了在运动过程中获得几种静脉血标本的有效方法。这种方法可用于收集样本, 可用于研究几个分子在运动期间 (例如, 代谢物, 荷尔蒙, 蛋白质和 RNA)。

身体活动过程中代谢和内分泌标志物的测量与理解不同运动方式的生理影响有关。在某些锻炼方式 (例如高强度间隔练习 (HIIE)) 中, 血液代谢物和荷尔蒙水平在相对较短的时间内波动1,2,3。
许多体育运动 (即,足球、篮球和橄榄球) 由低强度运动和/或被动期间与4、5之间的短时间高强度运动组成。HIIE 与上述运动相似, 因此被广泛用作运动员的训练方法6。此外, 由于其与运动的相似性, HIIE 已被用作研究运动生理学的模型7。采血是衡量生理变量的关键, 它经常被用于研究目的和身体状况的评估。然而, HIIE 期间的血液取样在技术上是困难的, 因为样品收集之间的时间很短。为解决这一问题, 我们在这里提出了一种方法来获得血液样本的 antecubital 静脉插管在短时间内, 而受试者执行 HIIE。

<table border="0" cellpadding="0" cellspacing="0" width="626">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Cycle ergometer</td>
			<td style="width:71px;">Monark</td>
			<td colspan="2" style="width:339px;">MONARK Ergomedic 874 E</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Catheter</td>
			<td style="width:71px;">Terumo</td>
			<td style="width:172px;">SR-FF2032</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Vacutainer</td>
			<td style="width:71px;">Terumo</td>
			<td>PZ-D03</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Tube for catheter</td>
			<td style="width:71px;">Terumo</td>
			<td style="width:172px;">SP-PTW30L02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heparin</td>
			<td style="width:71px;">Terumo</td>
			<td>PF-10HF10UA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lactate pro2</td>
			<td style="width:71px;">Arkray</td>
			<td>&#65324;&#65332;-1730</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulin ELISA kit</td>
			<td>Abnova</td>
			<td>ELISA Kit KA0921</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of blood lactate and plasma insulin during high-intensity exercise by antecubital vein catheterization

身体活动过程中代谢和内分泌标志物的测量与理解不同运动方式的生理影响有关。在一些锻炼方式 (例如,高强度间隔练习) 中, 血液代谢物和荷尔蒙水平在短时间内会发生变化。在本研究中, 我们描述了一种 catheterize antecubital 静脉的方法, 它允许在运动过程中采集几个血样。应用该方法对高强度运动中的胰岛素和静脉乳酸浓度进行测定。演习包括三三十年代的高强度运动, 由4分钟的恢复分离。在上一个恢复期之后, 执行了一个盖特测试。antecubital 静脉的血液样本是在三十年代前后, 以及在盖特试验前后获得的。因此, 在运动过程中, 可以对血浆胰岛素和静脉血乳酸的变化进行评价。

作为有代表性的结果, 我们使用该协议来测量在一个周期测力计 HIIE 中的六个实验对象的血乳酸和胰岛素的变化。盖特试验的峰值功率和平均功率分别为 732 120 和549瓦84瓦特。在三十年代 HIIE 期间, 平均功率用于计算功率输出 (494 瓦75瓦特)。
图 2显示了 HIIE 期间血浆胰岛素和血乳酸的变化。血浆胰岛素 (图 2A) 在三十年代的第一次和第三次发作期间减少, 但在第二回合和恢复期增加。另一方面, 血浆乳酸 (图 2B) 减少了在第二和第三三十年代回合, 但增加了在第一个三十年代回合和恢复期间。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56890/56890fig1.jpg"> 图 1: 练习协议.红色箭头表示采集血样的时间。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56890/56890fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56890/56890fig2.jpg"> 图 2: 实验结果.(A) 血浆胰岛素 (乌卢/mL)。(B) 血乳酸 (毫摩尔)。每种颜色代表一个不同的主题。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56890/56890fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization at JoVE.com

Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization

在这里, 我们提出了一个协议, 以获得几个血液样本从 antecubital 静脉在高强度间歇训练。该协议可能有助于测量血液代谢物和内分泌标记在运动期间。

Antecubital 静脉插管高强度运动时血乳酸和血浆胰岛素的评价

The measurement of metabolic and endocrinal markers during physical activity is of relevance to understanding the physiological implications of different ...

evaluation-blood-lactate-plasma-insulin-during-high-intensity

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

6.4K 次观看.  Kyoto University. 在这里, 我们提出了一个协议, 以获得几个血液样本从 antecubital 静脉在高强度间歇训练。该协议可能有助于测量血液代谢物和内分泌标记在运动期间。

视频: Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

补消耗的特定细胞群的枯竭

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

科研

免疫与感染

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

特定细胞群的净化荧光激活细胞分选（FACS）

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

手指棒运动诱导淋巴细胞凋亡的测定血液抽样方法

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

细胞迁移的的Phagokinetic轨道运动MLCK的定量评价

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

紧凑型超支化聚乙二醇接枝的膜抗原受保护的功能性红血细胞

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

对于血液处理快速的方法，以提高血浆肽水平的人血产量

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

由荧光相关光谱测量的一次淋巴细胞的细胞膜分子扩散

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...